Project description:Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying retinal vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we found that venous and arterial endothelial cells are in distinct cell cycle states during development and in adulthood. That is, venous endothelial cells reside in early G1 state, while arterial endothelial cells reside in late G1 state. Single cell RNA sequencing of developing retinal endothelial cells revealed that BMP signaling and early G1 state are enriched in venous endothelial cells, while TGF-b signaling and late G1 state are enriched in arterial endothelial cells. Cultured endothelial cells in early vs. late G1 exhibited significant differences in gene expression and activity, especially among BMP/TGF-b signaling components. The early G1 state was found to be essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevented the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.

Project description:Formation and maturation of a functional blood vascular system is required for the development and maintenance of all tissues in the body. During the process of blood vessel development, primordial endothelial cells are formed and become specified toward arterial or venous fates to generate a circulatory network that provides nutrients and oxygen to, and removes metabolic waste from, all tissues. Specification of arterial and venous endothelial cells occurs in conjunction with suppression of endothelial cell cycle progression, and endothelial cell hyperproliferation is associated with potentially lethal arterial-venous malformations. However, the mechanistic role that cell cycle state plays in arterial-venous specification is unknown. Herein, studying vascular development in Cdh5-CreERT2;R26FUCCI2aR reporter mice, we find that venous and arterial endothelial cells exhibit a propensity for different cell cycle states during development and in adulthood. That is, venous endothelial cells are predominantly FUCCI-Negative, while arterial endothelial cells are enriched for the FUCCI-Red reporter. Single cell RNA sequencing analysis of developing retinal endothelial cells reveals that venous endothelial cells are enriched for the FUCCI-Negative state and BMP signaling, while arterial endothelial cells are enriched for the FUCCI-Red state and TGF-b signaling. Further transcriptional analyses and live imaging of cultured endothelial cells expressing the FUCCI reporter show that reporter-negative corresponds to an early G1 state and reporter-red corresponds to late G1 state. We find the early G1 state is essential for BMP4-induced venous gene expression, whereas late G1 state is essential for TGF-b1-induced arterial gene expression. In a mouse model of endothelial cell hyperproliferation and disrupted arterial-venous specification, pharmacological inhibition of endothelial cell cycle prevents the vascular defects. Collectively, our results show that endothelial cell cycle control plays a key role in arterial-venous network formation, and distinct cell cycle states provide distinct windows of opportunity for the molecular induction of arterial vs. venous specification.

Project description:During embryonic vascular development, endothelial cells that line all blood vessels undergo specification into arterial, capillary, and venous subtypes to form a circulatory network. Regulation of endothelial cell cycle state has been shown to play a critical role in enabling arterial-venous specification and remodeling in a postnatal, blood flow-mediated, and tissue specific manner; however, if a similar cell cycle-mediated mechanism is a common mechanism that regulates embryonic vascular development, even prior to blood flow, is unknown. To investigate this, we first defined the emergence of distinct subtypes by isolating endothelial cells from wild type embryos at embryonic day (E)8.0, prior to blood flow; E8.5, just after flow begins; and E9.5. We performed single cell RNA sequencing and analyses, and found increased specification of arterial, venous and hemogenic subtypes over time, concomitant with decreased primordial and capillary endothelial cells. Gene Ontology analysis revealed that cell cycle control was significantly enriched over time, and we found arterial identity highly correlated with growth arrest. To gain more insight into the potential role of cell cycle control in specification, we isolated similar endothelial cells from Fucci-expressing embryos, sorted them into distinct cell cycle states (early G1, late G1 and S/G2/M), performed bulk RNA sequencing, and bioinformatically correlated endothelial cell cycle states with subtype identities. We found venous endothelial cells are highly enriched in early G1 and arterial endothelial cells highly enriched in late G1, which was corroborated with fluorescent imaging of Fucci embryos. Furthermore, we showed that endothelial cell hyperproliferation, induced by deletion of cell cycle inhibitor Cdkn1b (p27), impaired arterial-venous specification and vascular development. These results support that at the earliest stage of vascular development, endothelial cells in arterial and venous vessels reside in different cell cycle states and endothelial cell cycle control is required for their specification.

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Regulation of endothelial cell chromatin availability and transcription factor activity in arterial-venous specification - HUVEC FUCCI RNAseq

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